2001 Fiscal Year Final Research Report Summary
Studies on functions of mammalian RAD23 homologs that link DNA repair and protein degradation systems
| Project/Area Number |
12680680
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | RIKEN (The Institute of Physical and Chemical Reseach) |
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Principal Investigator |
SUGASAWA Kaoru RIKEN, Cellular Physiology Laboratory Senior Research Scientist, 細胞生理学研究室, 先任研究員 (70202124)
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| Project Period (FY) |
2000 – 2001
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| Keywords | nucleotide excision repair / xeroderma pigmentosum / XPC-HR23B complex / ubiquitylation / sumoylation |
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| Research Abstract |
To examine the possibility of ubiquitylation of the XPC protein that is specifically induced upon DNA damage, FLAG-tagged XPC (FLAG-XPC) and HA-tagged ubiquitin were simultaneously overexpressed in XP4PASV cells, which are derived from a XP group C patient and do not express endogenous XPC protein. By immunoprecipitation of FLAG-XPC from the cell extract, it was demonstrated that XPC can be indeed ubiquitylated in vivo. From similar experiments using various truncated XPC proteins, a domain in XPC was found, which is relatively more susceptible to the ubiquitylation. To investigate possible roles of the XPC ubiquitylation, we established a cell line that stably expresses a physiological amount of FLAG-XPC. When the cells were treated with DNA damaging agents such as UV and 4-NQO, a fraction of XPC became more tightly bound to chromatin, while the majority of ubiquitylated XPC could be solubilized under the same conditions. Moreover, we found that the XPC ubiquitylation occurred in a cell-cycle-independent manner, at least from the Gl/S boundary to the M phase.
To explore novel factors that interact with XPC, the yeast two-hybrid screening was carried out, which resulted in the ubiquitin-like protein SUM0-1 as well as the SUMO-1 conjugating enzyme Ubc9.As done for ubiquitylation, we demonstrated that XPC can be also sumoylated in cells by simultaneous overexpression of FLAG-XPC and HA-SUMO-1.
Using mouse embryonic fibroblast cells that lack both HR23A and HR23B, we established cell lines that stably express various mutant HR23B proteins. It was shown that the N-terminal ubiquitin-like domain of HR23B is essential neither for intracellular stabilization of XPC nor removal of UV-damage from the global genome.
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Research Products
(10 results)
